Document Xz36VnDnZ3VmjX48jJV697Y6J

To: Pruitt, Scott[Pruitt.Scott@epa.gov];Beck, Nancy[Beck.Nancy@epa.gov]; Bennett, Tate[Bennett.Tate@epa.gov]; Brown, Byron[brown.byron@epa.gov]; Jackson, RyanOackson.ryan@epa.gov] Cc: miehelle@epa.gov[michelle@epa.gov];Roy Bailey[rbailey@gdcille.com] From: Roy Bailey Sent: Sun 7/16/2017 10:28:10 PM Subject: Revised memo EPA Improperly Granted the Wolbachia EUF docx 0001.htm Administrator, I have attached a revised memo which we think may be even more compelling and helpful. ( sorry that it wasn't included in the previous email. ) We are available for a call anytime today or tomorrow that is convenient for your team. Again, we hope the EPA will consider revoking the field trial permit EUP for the referenced technology until the EPA can complete a full impact study on humans and the environment. Thanks for your interest and consideration. Respectfully and best regards Roy W. Bailey CEO Giuliani Deason Capital Interests, LLC Cell 214.208.1721 Office 214.378.3663 Rbailey@gdcillc.com Rbailev@bailevstrategicadvisors.com Begin forwarded message: From: "Bobo, Jack" <JBobo@intrexon.com> Date: July 16, 2017 at 3:07:45 PM PDT To: 'Roy Bailey' <rbailev@gdcillc.com> Subject: EPA Memo Updated CONFIDENTIAL TRANSMISSION - To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003007-00001 Intrexon Corporation to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. 17cv1906 Sierra Club v. EPA 6/22 Production ED 001523 00003007-00002 EPA Must Revoke the Improperly Granted Wolbachia Aedes aegypti Experimental Use Permit EPA's Office of Pesticide Programs improperly granted an Experimental Use Permit (EUP) to MosquitoMate that permits the release of millions of Wolbachia-infcctcd Aedes aegypti mosquitoes in California and Florida [1], Oxitec and other commenters submitted substantive comments on the record identifying significant concerns and critical risks associated with the proposed EUP. Notwithstanding these comments, EPA granted the requested EUP to MosquitoMate. Subsequent to EPA's improvident grant of approval for the release of millions of Wo/bachia-infectcd Aedes aegypti new data and information has become available that highlights to an even greater extent the potential risks of such releases. EPA can immediately remedy this improper regulatory approval and all other current Wolbachia EUPs. Pursuant to FIFRA Section 5(e) (7 U.S.C. 136c(e)), the EPA Administrator may revoke an EUP immediately if he finds that its terms or conditions are being violated or that its terms or conditions are inadequate to avoid unreasonable adverse effects on the environment. (See also 40 C.F.R. 172.10(a)) EPA's regulations provide that a permittee may "contest" the notice of revocation by requesting "an opportunity to confer with the Administrator." The Administrator will issue a final decision within 20 days after such conference. (40 C.F.R. 172.10(c)) Neither FIFRA nor EPA's regulations provide for a formal hearing to object to revocation of an EUP. EPA's granting of an experimental use permit to MosquitoMate for an Aedes aegypti artificially infected with Wolbachia pipientis was premature and did not properly assess the potential adverse impacts to the environment that may result from releases of Aedes aegypti artificially infected with a bacterium that is not native to the Aedes aegypti microbiome. EPA did not conduct a necessary rigorous assessment of the potential adverse impacts to human health and the environment that could result from uncontained release of Aedes aegypti containing this non native strain of bacteria. Wolbachia is a complex bacterium that is known to biologically alter its hosts. [5] Wolbachia is found in many different insects in nature. Yet that does not make its artificial insertion into Aedes aegypti "natural" or "safe". Serious and very basic questions remain as to the potential adverse threats that Wolbachia poses to human health and the environment. In cases where Wolbachia inhabits insects that bite humans such as mosquitoes and flies, numerous adverse effects have been documented in peer-reviewed publications. Therefore, using Wolbachia as a mosquito control solution likely presents very significant risks to public health and the environment. Some, but by no means all, of the serious health, safety, and environmental concerns regarding Wolbachia, include: Wolbachia holds several key roles in River Blindness which begins with a bite of a blackfly. River Blindness infects up to 25 million people globally and is the second most common infectious cause of blindness; [16] Wolbachia has been attributed to major lymphatic inflammation associated with Elephantiasis, also known as lymphatic filariasis, which is spread by the bites of infected 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00001 mosquitoes; [15, 17] Wolbachia significantly enhances West Nile virus infection in mosquitoes increasing risk of transmission to humans; [8] Wolbachia enhances malaria parasite infection in mosquitoes increasing risk of transmission to humans; [7, 9, 10, 12] and Wolbachia has been naturally found in certain mosquitoes (not artificially inserted in a lab), and those infected mosquitoes still are able to transmit dangerous viruses such as dengue and chikungunya. [11] In addition to these direct and dangerous threats to humans, Wolbachia transfers genes to its hosts through a process called Horizontal Gene Transfer (HGT). [2, 3, 4, 5] Thus, artificially infecting Aedes aegypti with Wolbachia can effectively introduce over 1,000 new genes into its mosquito hosts (as compared to recombinant genetic engineering which typically introduces a few genes or less). This random genetic engineering is not well-defined or understood, yet the result is that every time Wolbachia invades a host, or is artificially inserted into a host mosquito, the result is an undefined, randomly genetically modified organism. This stands in stark contrast to precisely genetically engineered organisms, where the genetic transformation is well defined and well understood. Moreover, it appears that Wolbachia may be capable of mediating significant lateral gene transfer to higher organisms. [5, 6, 13] Peer review papers show that Wolbachia genetically modifies its hosts as it transfers its DNA into insect genomes and these genes are expressed; Wolbachia have high rates of natural DNA genetic change (recombination); [18] and, Of significant concern is newly published evidence confirming that Wolbachia has a virus that encodes and causes the expression of a toxin from the Black widow spider that is hypothesized to form pores in cell membranes. If mosquitoes that carry Wolbachia with this virus are released, the effects of this virus are unknown as are the consequences of these mosquitoes biting people. [14] Appended is a list of some of the recent publications that provide data and information that raise significant concerns as to the serious adverse public health and environmental effects that may result from release of Wolbachia-infected mosquitoes. Some of these effects were well known at the time that EPA granted the Wolbachia Aedes aegypti EUP. Others constitute new information regarding potential adverse effects of release of these mosquitoes. EPA should have, but did not, adequately evaluate the known adverse effects of Wolbachia at the time the EUP was granted. Moreover, the more recent data and information substantially increase concerns regarding the potential release of millions of Wolbachia Aedes aegypti. To date, MosquitoMate has not released large numbers of the Wolbachia Aedes aegypti under the approved EUP. However, as is demonstrated in the published literature referenced below, and as Oxitec and other commenters demonstrated on the record of the EUP application, releases of such massive numbers of these artificially infected Aedes aegypti could result in serious unreasonable adverse effects to public health and the environment. MosquitoMate must not be allowed to risk these potentially significant adverse effects to public health and the environment. 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00002 References 1. USEPA, Amendments, Extensions, and/or Issuances of Experimental Use Permits, 81 Federal Register 69059 October 5, 2016. 2. Woolfit, M., et al.. An ancient horizontal gene transfer between mosquito and the endosymbiotic bacterium Wolbachiapipientis. Molecular biology and evolution, 2009. 26(2): p. 367-74. 3. Horizontal gene transfer between Wolbachia and the mosquito Aedes aegypti. https://www.ncbi.nlm.nih.gov/pubmed/19154594 Excerpt - The evolutionary importance ofhorizontal gene transfer (HGT) from Wolbachia endosymbiotic bacteria to their eukaryotic hosts is a topic ofconsiderable interest and debate...We have discovered a case ofHGT, involving two adjacent genes, between the genomes of Wolbachia and the currently Wolbachia-uninfected mosquito Aedes aegypti, an important human disease vector...The data support the argument that HGT between Wolbachia endosymbiotic bacteria and their hosts has produced evolutionary innovation. Authors - Klasson L, Kambris Z, Cook PE, Walker T, Sinkins SP 4. A case of horizontal gene transfer from Wolbachia to Aedes albopictus C6/36 cell line a. https://www.ncbi.iilm.nih.gov/pmc/articles/PMC40.13104/ Excerpt - Horizontal gene transfer plays an essential role in evolution and ecological adaptation, yet this phenomenon has remained controversial, particularly where it occurs between prokaryotes and eukaryotes. In this study, we report the discovery of a horizontal gene transferfrom the endosymbiont Wolbachia in the C6/36 cell line derivedfrom the mosquito Aedes albopictus. Moreover, we report that this horizontally transferred gene displayed high transcription level. This finding and the results of further experimentation strongly suggest this gene is functional and has been expressed and translated into a protein in the mosquito host cells. Authors - Qing Hou , Ji He, Jing Yu, Yuting Ye, Dan Zhou, Yan Sun, Donghui Zhang, Lei Ma, Bo Shen, and Changliang Zhu 5. Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. a. https://www.ncbi.nlm.nih.gov/pubme -8 Excerpt - Although common among bacteria, lateral gene transfer, i.e., the movement ofgenes between distantly related organisms, is thought to occur only rarely between bacteria and multicellular eukaryotes. However, the presence ofendosymbionts, such as Wolbachia pipientis, within some eukaryotic germlines may facilitate bacterial gene transfers to eukaryotic host genomes. Wefound and confirmed transfers into the genomes offour insect andfour nematode species that rangefrom nearly the entire 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00003 Wolbachia genome (>1 megabase) to short (<500 base pairs) insertions. Authors - Dunning Hotopp JC, Clark ME, Oliveira DC, Foster JM, Fischer P, Munoz Torres MC, Giebel JD, Kumar N, Ishmael N, Wang S, Ingram J, Nene RV, Shepard J, Tomkins J, Richards S, Spiro DJ, Ghedin E, Slatko BE, Tettelin H, Werren JH. 6. Genome fragment of Wolbachia endosymbiont transferred to X chromosome of host insect a. http://www.pnas.org/content/99/22/.14280.ful1 Excerpt - Here we report an unprecedented case ofprokaryote-eukaryote horizontal gene transfer: a genomefragmentfrom the Wolbachia endosymbiont has been transferred to the X chromosome ofa beetle.... The adzuki bean beetle, Callosobruchus chinensis, is triple-infected with distinct lineages of Wolbachia endosymbiont, wBruCon, wBruOri, and wBruAus, which were identified by their wsp (Wolbachia surface protein) gene sequences. Whereas wBruCon and wBruOri caused cytoplasmic incompatibility ofthe host insect, wBruAus did not. Although wBruCon and wBruOri were easily eliminated by antibiotic treatments, wBruAus persisted over five treated generations and could not be eliminated...The study's results strongly suggest that wBruAus has no microbial entity but is a genome fragment of Wolbachia endosymbiont transferred to the X chromosome ofthe host insect. Authors - Natsuko Kondo, Naruo Nikoh, Nobuyuki Ijichi, Masakazu Shimada, and Takema Fukatsu 7. Wolbachia Can Enhance Plasmodium Infection in Mosquitoes: Implications for Malaria Control? http://ioumals.plos.org/plospathogens/article?id=.10. .1.37.1/journal .opal.1004.182 Excerpt - Any potential control strategy devised in regions where more than one parasite species occurs needs to thoroughly investigate the effect of Wolbachia on all parasite species transmitted by the vector, as well as other pathogens such as filarial worms or arboviruses to ensure that Wolbachia-infected mosquitoes do not inadvertently enhance transmission ofsecondary pathogens. Authors - Grant L. Hughes, Ana Rivero, Jason L. Rasgon 8. Wolbachia Enhances West Nile Vims (WNV) Infection in the Mosquito Culex tarsalis a. http://ioumals.plos.org/plosntds/article?id=.10..137.1 /iournal.pntd.0002965 Excerpt - This is thefirst observation ofWoI bac hia-/7?duced enhancement ofa human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part ofa vector-borne disease controlprogram. Authors - Brittany L. Dodson, Grant L. Hughes, Oluwatobi Paul, Amy C. Matacchiero, Laura D. Kramer, Jason L. Rasgon 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00004 9. Wolbachia increases susceptibility to Plasmodium infection in a natural system a. http://rspb.rovalsocietYpublishing.Org/con.ten.t/281/1779/20.132837 Excerpt - These results suggest that naturally Wolbachia-infected mosquitoes may, in fact, be better vectors ofmalaria than Wolbachia-free ones. Authors - F. Zele, A. Nicot, A. Berthomieu, M. Weill, O. Duron, A. Rivero 10. Wolbachia Strain wAlbB Enhances Infection by the Rodent Malaria Parasite Plasmodium berghei in Anopheles gambiae Mosquitoes a. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294472/ Excerpt - Wolbachia, a common bacterial endosymbiont ofinsects, has been shown to protect its hosts against a wide range ofpathogens. However, not all strains exert a protective effect on their host. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen... Authors - Grant L. Hughes, Joel Vega-Rodriguez, Ping Xue, and Jason L. Rasgon 11. Wolbachia in a major African crop pest increases susceptibility to viral disease rather than protects. a. https://www.ncbi.nlm.nih.gov/pubmed/22731846 Excerpt - Wolbachia have generated considerable recent interest due to the capacity of some strains to protect their insect hosts against viruses and the potentialfor this to reduce vector competence ofa range ofhuman diseases, including dengue. In contrast, here we provide datafrom fieldpopulations ofa major crop pest, African armyworm (Spodoptera exempta), which show that the prevalence and intensity ofinfection with a nucleopolydrovirus (SpexNPV) is positively associated with infection with three strains of Wolbachia Authors - Graham RI, Grzywacz D, Mushobozi WL, Wilson K. 12. Temperature alters Plasmodium blocking by Wolbachia. a. https://www.ncbi.nlm.nih.gov/pubmed/24488176 Excerpt - Very recently, the Asian malaria vector (Anopheles stephensi) was stably transinfected with the wAlbB strain of Wolbachia, inducing refractoriness to the human malaria parasite Plasmodium falciparum. However, conditions in thefield can differ substantially from those in the laboratory. Our results demonstrate complex 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00005 effects oftemperature on the Wolbachia-malaria interaction, and suggest the impacts oftransinfection might vary across diverse environments. Authors - Murdock CC, Blanford S, Hughes GL, Rasgon JL, Thomas MB. 13. Phylogenetic relationships of the Wolbachia of nematodes and arthropods. a. https://www.ncbi.nlm.nih.gov/pubmed/17040125 Excerpt - Using the wOvo sequence, we identified a lateral transfer event whereby segments of the Wolbachia genome were inserted into the Onchocerca nuclear genome. This eventpredated the separation ofthe human parasite O. volvulus from its cattle-parasitic sister species, O. ochengi. The long association between filarial nematodes and Wolbachia symbionts may permit more frequent genetic exchange between their genomes. Authors - Fenn K, Conlon C, Jones M, Quail MA, Holroyd NE, Parkhill J, Blaxter M 14. Eukaryotic association module in phage WO genomes from Wolbachia a. http://www.nature.com/articles/ncommsl 3155 Excerpt - Here we report a metagenomic analysis ofpurified bacteriophage WO particles of Wolbachia and uncover a eukaryotic association module in the complete WO genome. It harbours predicted domains, such as the black widow latrotoxin Cterminal domain, that are uninterrupted in bacteriophage genomes, enriched with eukaryotic protease cleavage sites and combined with additional domains to forge one ofthe largest bacteriophage genes to date (14,256 bp). To the best ofour knowledge, these eukaryotic-like domains have never before been reported in packaged bacteriophages and their phytogeny, distribution and sequence diversity imply lateral transfers between bacteriophage/prophage and animal genomes. Finally, the WO genome sequences and identification ofattachment sites willpotentially advance genetic manipulation of Wolbachia. Authors - Sarah R. Bordenstein & Seth R. Bordenstein 15. Onchocerciasis: the Role of Wolbachia Bacterial Endosymbionts in Parasite Biology, Disease Pathogenesis, and Treatment a. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131055/ Excerpt - Studies with otherfilarial nematode species have also highlighted a rolefor Wolbachia in transmission and infection of the mammalian host through a fascinating manipulation ofmast cell-mediated vasodilation to enhance infectivity ofvector-borne larvae. Wolbachia has also been identified as the principal driver ofinnate and adaptive Thl inflammatory immunity, which can either contribute to disease pathogenesis or, with the Wolbachia-mediated recruitment ofmast cells, enhance infectivity. The Wolbachia activation ofinnate inflammation also drives inflammatory adverse events in response to chemotherapy with either diethylcarbamazine (DEC) or 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00006 ivermectin. In this review we summarize the experimental andfield trial data which have uncovered the importance of Wolbachia symbiosis in onchocerciasis. Authors - 16. The Role of Endosymbiotic Wolbachia Bacteria in the Pathogenesis of River Blindness a. http://www.nature.eom/news/2002/020304/full/news020304-9.html Excerpt - Using a murine modelfor river blindness in which soluble extracts offilarial nematodes were injected into the corneal stroma, we demonstrated that the predominant inflammatory response in the cornea was due to species to endosymbiotic Wolbachia bacteria. In addition, the inflammatory response induced by these bacteria was dependent on expression offunctional Toll-like receptor (TLR4) on host cells. Authors - Amelie v. Saint Andre, Nathan M. Blackwell, Laurie R. Hall, Achim Hoerauf, Norbert W. Brattig, Lars Volkmann, Mark J. Taylor, Louise Ford, Amy G. Hise, Jonathan H. Lass, Eugenia Diaconu, Eric Pearlman 17. Wolbachia bacteria in filarial immunity and disease. a. https://www.nebi.nlm.nih.gov/pubmed/.1.1472559 Excerpt - Lymphatic filarial nematodes are infected with endosymbiotic Wolbachia bacteria. Lipopolysaccharidefrom these bacteria is the major activator ofinnate inflammatory responses induced directly by the parasite. Here, we propose a mechanism by which Wolbachia initiates acute inflammatory responses associated with death ofparasites, leading to acutefilarial lymphangitis and adverse reactions to antifilarial chemotherapy. We also speculate that repeated exposure to acute inflammatory responses and the chronic release ofbacteria, results in damage to infected lymphatics and desensitization ofthe innate immune system. These events will result in an increased susceptibility to opportunistic infections, which cause acute dermatolymphangitis associated with lymphoedema and elephantiasis Authors - Taylor MJ, Cross HF, Ford L, Makunde WH, Prasad GB, Bilo K. 18. Comparative Genomics of Wolbachia and the Bacterial Species Concept, https://www.nebi.nlm.nih.gOv/pmc/artic1es/P 33/ Excerpts - The importance ofhost-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability ofrecombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. Finally, we inferred several cases ofgene transfers ofphages between strains of different supergroups. All genomes contain prophages ofsimilar organization IFigure 851 and our single gene phytogenies ofphage genes revealed a mosaic pattern, consistent with repeated recombination ofphage genes within and across the 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00007 supergroup boundary. 17cv1906 Sierra Club v. EPA - 6/22 Production ED 001523 00003009-00008